RU2692785C1 - Methods of reducing cachexia associated with cancer - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions relates to medicine and a method of reducing cancer induced cachexia in a mammal, comprising administering class 1 and 2b HDAC inhibitor to said mammal in an amount effective to maintain a mammalian body weight, compared to a mammal that does not receive HDAC class 1 and 2b inhibitor. Group of inventions also relates to a method of supporting the skeletal muscle mass in a mammal having cancer, where the skeletal muscle mass is reduced by cancer, involving administering class 1 and 2b HDAC inhibitor to said mammal.

EFFECT: group of inventions provides reduced cancer-induced cachexia.

9 cl, 10 ex, 11 dwg

Description

2420-534633RU / 042

PRIORIZED PRIORITY

This application claims priority on provisional patent application US registration No. 61/906738, filed November 20, 2013. The above application is included in the present description by means of, which is equivalent to its full content. All references cited in the present description, including, without limitation, patents and patent applications, are incorporated by reference in their entirety.

PRIOR ART

Cachexia associated with cancer is a weakened condition associated with loss of muscle mass, fatigue, weakness and loss of appetite. Cachexia is also associated with severe clinical consequences, including muscle weakness, which can lead to difficulty in movement and pulmonary complications. Cachexia is an essential factor contributing to the death of cancer patients.

Cachexia is characterized by loss of skeletal muscle mass, which is not amenable to reverse development with normal nutritional support, leading to pronounced loss of body mass, which significantly affects the morbidity and mortality of patients. It develops in more than 80% of patients with cancer of the stomach, pancreas, and esophagus; 70% of those with head and neck cancer; and approximately 60% of patients with lung cancer, colorectal cancer and prostate cancer. See, for example, Muscle (2012) 3, 245-51. Despite the effect of cachexia on mortality among patients with cancer, no effective therapy has been developed to prevent or deter the progression of cachexia. For example, it is estimated that more than 85% of patients with pancreatic cancer, including patients with early stages, lose an average of 14% of their body weight before the disease. See, for example, BMC Cancer. 2010 Jul 8; 10: 363. Cachectic patients with pancreatic cancer are often weak and tired, and have lower tolerance to therapy and more undesirable outcomes of operations. Consequently, cachexia is the leading cause of death in pancreatic cancer. Unfortunately, the 5-year survival rate for pancreatic cancer has remained at 6% over the past four decades, which is the smallest among all malignant tumors.

With the introduction of new means for identifying cachectic factors and their effects on skeletal muscle in the area of cachexia, significant advances have recently been made in understanding the underlying mechanisms that regulate muscle atrophy in cancer and other chronic diseases. As a result, the authors now understand how cytokines and systemic inflammation regulate muscle atrophy by acting on key signaling pathways that act from inside the myofiber. However, translation of the discoveries obtained into effective treatment was difficult, and there was no effective treatment for cachexia to the aspects described in the present description.

The skeletal muscle mass is regulated, in part, by the relative rate of protein synthesis compared to the regulation of protein. Alamdari, N, et al., Acetylation and deacetylation — novel factors in muscle wasting, Metabolism. Jan 2013; 62 (1): 1-11. The loss of skeletal muscle mass develops when the rate of protein breakdown is greater than protein synthesis. Ibid. Acetylation and deacetylation of the protein modifies the transcription factors and the transcription of genes that can affect muscle mass, making proteins more or less susceptible to degradation. Ibid. Histone acetylases (HAT) and histone deacetylases (HDAC) play a role in the regulation of protein acetylation and deacetylation.

However, the effects of such molecules on muscle loss and cachexia were contradictory — evidence suggests, for example, that the use of HDAC inhibitors (for example, Trichostatin A (TSA)) leads to hyper-acetylation, which can stimulate protein degradation, leading to increased muscle loss and cachexia. Conflicting results were found by Narver et. al., (Sustained improvement of spinal muscular atrophy mice treated with trichostatin A plus nutrition. Ann Neurol. 2008; 64: 465-70) however, the results were questionable, since the treatment of TSA was also accompanied by aggressive nutritional support. Alamdari et al., In 5. Therefore, it is believed that HDAC inhibitors, rather than enhance, reduce cachexia, or their use gave conflicting and contradictory results.

The development and progression of cancer cachexia is caused by complex, multifactorial pathophysiological responses to muscle tissue tumors. FDA-approved treatment is currently unavailable to prevent or slow the progression of muscle loss in patients with cachexia. To date, several investigational drugs that target various aspects of the pathogenesis of cachexia are being studied in humans, however, with different clinical outcomes. For example, whereas mAb BYM38 from Novartis (bimagrumab), which blocks the binding of myostatin and activin to receptors of activin type II, was given the FDA designation as breakthrough therapy, a drug for loss of muscle GTx's enobosarm, a selective androgen receptor modulator, was not included in clinical studies late stage.

Acetylation of nuclear histones plays an important role in the regulation of gene transcription by regulating nucleosomal DNA packaging. Histone deacetylation leads to precise nucleosome packaging and transcriptional repression due to the limited access of transcription factors to DNA targets. Acetylation of histones relaxes the structure of nucleosomes, providing greater access for transcription factors. The balance between deacetylation and acetylation of histones is modulated by histone deacetyltransferase (HDAC) and histone acetyltransferase (HAT). The pathological balance of such factors correlates with the growth of pathological cells and some forms of cancer, as discussed in US patent number 8318808, incorporated by reference in its entirety in the present description. HDAC inhibitors, in particular, changes in the balance between acetylation and deacetylation, leading to stunting, differentiation and apoptosis in many types of tumor cells. See, for example, US Patent Number 8318808.

18 HDACs have been identified in humans and are characterized as being zinc-dependent or nicotinamide adenine dinucleotide (NAD) -dependent (Discov Med 10 (54): 462-470, November 2010) and associated with the following classes: class I (HDAC 1, 2 , 3, and 8); class II (HDAC 4, 5, 6, 7, 9, and 10; class III (sirtuins 1-7 (SIRT)); and class IV (HDAC 11).

Among them, of particular interest are inhibitors of HDAC, described in US patent number 8318808, and based on, for example, fatty acids associated with Zn ²⁺ -complexing fragments through aromatic Ω-amino acid linkers. In various aspects, HDAC inhibitors may have the formula:

where X is selected from H and CH3; Y is (CH2) n, where n is 0-2; Z is selected from (CH2) m, where m is 0-3 and (CH) 2; A is a hydrocarbyl group; B represents an o-aminophenyl or hydroxyl group; and Q is halogen, hydrogen, or methyl. One HDAC inhibitor, in particular (N-hydroxy-4- (3-methyl-2-phenylbutyrylamino) benzamide) is also known as AR-42. In one aspect, the structure of the AR-42 is as follows:

AR-42 is a broad-spectrum deacetylase and histone and nonhistone protein inhibitor with demonstrated greater efficacy and activity in solid tumors and hematological malignant tumors when compared with vorinostatom (i.e., SAHA). See, for example, Lu YS, et al., Efficacy of a novel histone deacetylase inhibitor in murine models of hepatocellular carcinoma, Hepatology. 2007 Oct; 46 (4): l 119-30; Kulp SK, et al., Antitumor effects of a novel phenylbutyrate-based histone deacetylase inhibitor, (S) -HDAC-42, in cancer prevention, Clinical Cancer Res. 2006 Sep 1; 12 (17): 5199-206.

AR-42 may also have additional histone-independent mechanisms that are involved in its therapeutic profile. See, for example, Chen MC, et al., Novel mechanism by which histone deacetylase inhibitors facilitate topoisomerase α degradation in hepatocellular carcinoma cells, Hepatology. 2011 Jan; 53 (l): 148-59; Chen CS, et al., Histone acetylation-independent effect of histone deacetylase inhibitors at Junction, J Biol Chem. 2005 Nov 18; 280 (46): 38879-87; Yoo CB, et al., Epigenetic Therapy of Cancer: past, present and future, Nat Rev Drug Discov. 2006 Jan; 5 (l): 37-50.

AR-42 has a demonstrated inhibitory effect in tumors, including, without limitation, the mammary gland, prostate gland, ovaries, blood cells (for example, lymphoma, myeloma, and leukemia), the liver, and the brain. See, for example, Mims A, et. al., Increased anti-leukemic activity of decitabine via AR-42-induced upregulation of miR-29b: a novel epigenetic targeting approach, myeloid leukemia, Leukemia. 2012 Nov 26. doi: 10.1038 / leu.2012.342. [Epub ahead of print]; Burns SS, et al., Histone deacetylase inhibitor AR-42 differentially affects cell-cycle transient in meningeal and meningioma cells, potently inhibiting NF2-deficient meningioma growth, Cancer Res. 2013 Jan 15; 73 (2): 792-803; Lu YS, et. al., Radiosensitizing effect of a phenylbutyrate-derived histone deacetylase inhibitor in hepatocellular carcinoma, Int J Radiat Oncol Biol Phys. 2012 Jun 1; 83 (2); Zimmerman B, et. al., T-lymphotropic virus type 1 adult T cell car cell, Leuk Res. 2011 Nov; 35 (l l): 1491-7; Zhang S, et al., AR-42, inhibits gpl30 / Stat3 pathway and apoptosis cell cycle and arrest in multiple myeloma cells, Int J Cancer. 2011 Jul 1; 129 (1): 204-13.

SUMMARY OF INVENTION

The aspects presented herein provide methods for suppressing cachexia in a mammal with cancer, comprising administering a class 1 and 2 HDAC inhibitor to the said mammal. One aspect provides a method for suppressing cachexia in a mammal with cancer by administering an HDAC class 1 and 2b inhibitor to a specified mammal in an amount effective to substantially maintain the mass of the mammal as compared to a mammal that does not receive a class 1 and 2 HDAC inhibitor. In another aspect, an HDAC class 1 and 2b inhibitor is administered to a mammal with cancer in an amount effective to substantially maintain at least about 90% of the body weight of said mammal for a period of at least fifteen days. In another aspect, the HDAC class 1 and 2b inhibitor is AR-42. In another aspect, a mammal with cancer has at least one tumor and the tumor volume does not decrease by more than 6% for about the first fifteen days after AR-42 treatment.

Other aspects presented herein provide methods for suppressing cachexia by administering AR-42 to a mammal with cancer, where the expression of multiple mediators of muscle atrophy (eg, pro-cachexia stimulants, such as IL-6, IL-6RCC, LIF, MuRFl, atrogin -I) in cancer cachexia is reduced compared to a mammal with cancer that does not receive AR-42 treatment.

Additional aspects provide methods for suppressing cachexia by administering AR-42 to a mammal with cancer, where cachexia-induced fat loss and reduction in the size of musculoskeletal fibers are essentially restored compared to a mammal that does not receive AR-42.

Aspects described herein provide methods for maintaining skeletal muscle mass in a mammal having cancer by administering an HDAC class 1 and 2b inhibitor to said mammal in an amount effective to maintain at least about 90% of the skeletal muscle mass of said mammal for a time period of at least fifteen days compared to a mammal that does not receive a class 1 and 2b HDAC inhibitor.

Additional aspects provide methods for extending the survival of a mammal having cancer by administering an HDAC class 1 and 2b inhibitor to a mammal in an amount effective to essentially prolong the survival of the mammal compared to a mammal that does not receive a class 1 and 2b HDAC inhibitor.

As described in the present description, AR-42 demonstrates efficacy in vivo in suppressing, reducing, or blocking muscle loss and prolonging survival in animal cancer cachexia models. In addition, the effect of AR-42 on cancer-related cachexia does not depend on the effects of AR-42 on reducing the tumor burden.

BLUEPRINTS

FIG. Figure 1A shows an exemplary suppression of cancer-induced cachexia in mice with C-26 tumors, and shows changes in total mass (left, tumor is included) and body weight {in the center, tumor is excluded) during a 15-day study in mice without tumors receiving carrier (control), compared with mice with tumors receiving the carrier (carrier), or oral AR-42 at a dose of 50 mg / kg every other day (AR-42). The arrows indicate the treatment time of the AR-42. On the right, the lack of a suppressive effect of AR-42 on tumor growth in mice with C-26 tumors. Data are presented as mean ± S.D. P values: a, 0.045; b, 0.0027; c, 0.049; d, 0.0048;

FIG. Figure 1B shows photographs of characteristic mice with tumors from each group, depicting the therapeutic effect of AR-42 on cancer cachexia;

FIG. Figure 1C shows the approximate average daily food intake among the three treatment groups during the study. Data are presented as mean ± S.D. (n = 8);

FIG. 1D shows the approximate effects of AR-42 on muscle mass in the hind limbs, including the gastrocnemius, anterior tibialis and quadriceps (P values: a, <0.001; b, 0.0042; c, 0.0046) in mice without tumors and with tumors compared with those receiving the host mice with and without tumors (n = 8);

FIG. 1E shows the approximate effects of AR-42 on the heart, adipose tissue and spleen (P values: a, <0.001; b, 0.0059; c, 0.001; d, 0.009) in mice with and without tumors compared with those receiving mice with tumors and without tumors (n = 8);

FIG. Figure 2A shows an exemplary preservation of muscle fiber size in mice with a C-26 tumor, shown on the left, micrographs of H & E-stained sections of gastrocnemius muscles from control mice without tumors, and mice with tumors treated with vehicle or AR-42. The bars of the scale, 100 μm and on the right, the cross-sectional area of muscle fibers in the calf muscles, are presented as a histogram of frequency with certainty (P <0.001). Data are presented as mean ± S.D .;

FIG. 2B shows exemplary Kaplan-Meier survival curves for mice with tumors treated with vehicle, vorinostat (50 mg / kg, p.o., daily), romidepsin (0.6 mg / kg, and / p, twice a week), or AR-42 (50 mg / kg bp, every other day). Survival was determined at the time point in which the loss of body weight (excluding a tumor) reached 20% of the initial body weight, which served as a human end point for exclusion from the study (*, P <0.001, carrier vs. AR-42; n = 8);

FIG. Figure 2C shows exemplary photographs of characteristic mice from each group, depicting the therapeutic effect of AR-42 on vorinostat and romidepsin on cancer cachexia in mice with tumors, assessed by posture, coat and body condition;

FIG. 2D shows the approximate relative expression level of Atrogin-1 // MAFbx and MuRFl mRNA in skeletal muscles of mice without tumors treated with vehicle (n = 6) and mice with tumors treated with AR-42 (n = 8), vorinostat (n = 8 ), or romidepsin (n = 5) compared with that of mice with tumors treated with the carrier (n = 8) 15 days after injection of tumor cells. Data are presented as mean + S.D. P values: a, <0.001; b, 0.016; c, 0.0063;

FIG. 3A shows exemplary effects on the level of intermediates associated with glycolysis and alternative glucose metabolism pathways;

FIG. Figure 3B shows the approximate effects of AR-42 on glycogen metabolism in the calf muscles of mice with and without tumors (n = 8 for each group). Mice with tumors received vehicle or AR-42 (50 mg / kg, p.o., every other day) starting on day 6 after injection of tumor cells and ending on day 17. Control mice without tumors received carrier or AR-42 in parallel. The data are presented in blocks and graphs with scatter. The bottom and top of the bar represent the first and third quartiles and the “+” symbol and the bar outside the bar represent the mean and median, respectively. The ends of the whiskers represent the maximum and minimum values in each group;

FIG. 4 shows that AR-42 blocks changes in cachexia in the level of free amino acids and amino acid metabolites involved in the regulation of neurotransmission and biomarkers of insulin resistance in muscle mice with C-26 tumors. Samples for analysis were obtained from the experiment described in relation to FIG. 3 A and 3B. The data is presented in a “mustache box” diagram, as described in relation to drawings 3A and 3B;

FIG. 5A shows the approximate (top) effects of AR-42 on the level of cytokines for pro-cachexia IL-6 (left) and LIF (right) in the serum of mice without tumors compared to mice with C-26 tumors, and (bottom) qPCR analysis of the effects of AR -42 per expression of IL-6Ra mRNA in skeletal muscle of mice without tumors compared with mice with C-26 tumors. Data are presented as mean ± S.D. P values: a, <0.001; b, 0.006; c, 0.012 (n = 3). Mice were treated as described in relation to FIG. 3;

FIG. Figure 5B shows an exemplary analysis of the data components of RNA-seq (left) and a Venn diagram (right) showing differentially expressed genes among the four treatment groups. TF, no tumors; T, with tumors; veh receiving media; AR receiving AR-42. Mice were treated as described in relation to FIG. 3;

FIG. 5C shows an exemplary analysis of the effects of AR-42 on the transcript level of six key regulators of pro-cachexia in RNA-seq (P values: a, 0.024; b, 0.028; c, 0.015; d, 0.007; e, 0.024; f, 0.026; g , 0.01; h, 0.012; i, <0.001; j, 0.014; n = 3). Mice were treated as described in relation to FIG. 3;

FIG. 5D shows an exemplary analysis of the effects of AR-42 on transcript levels of six key regulators of pro-cachexia through qPCR in skeletal muscle in four treatment groups (*, P <0.001; n = 6). Data are presented as mean ± S.D. Mice were treated as described in relation to FIG. 3;

FIG. 6A shows suppression of cancer cachexia in mice bearing C-26 tumors by delaying AR-42 treatment to late stages of tumors and cachexia. Changes in body weight (with the exception of the tumor) during the 18 day study in control mice without tumors treated with vehicle (T / F, Veh) and mice with tumors treated with vehicle (T, Veh) compared with those treated orally AR-42 (shown on the left), starting on day 6 (T, AR42 / D6), day 10 (T, AR42 / D10), or day 12 (T, AR42 / D12). P values: a, 0.0015; b, 0.023 (n = 8). The arrows show the time points for the initiation of AR-42 treatment. Data are presented as average. For clarity of presentation, S.D. the bars for each data point are not shown. On the right, results showing the absence of a suppressive effect of AR-42 on tumor growth in mice with C-26 tumors in a delayed treatment experiment. Data are presented as mean ± S.D. (n = 8);

FIG. 6B shows exemplary photographs depicting the therapeutic effect of AR-42 on cancer cachexia in mice with tumors, despite deferred treatment, which manifests itself in normal posture, smooth hair, and better body condition, despite the greater tumor burden;

FIG. 6C shows the exemplary effects of AR-42 treatment starting at different stages of disease progression, as depicted in FIG. 6A, on the hindlimb muscle mass, including the gastrocnemius, anterior tibial and quadriceps, in mice with C-26 tumors. Data are presented as mean ± S.D. (n = 8; *, P <0.001);

FIG. 6D shows the effects of AR-42 on the compression force of mice with tumors relative to a control without tumors and with tumors receiving media on day 15 and day 16. Data are presented as mean ± S.D. (n = 8; P values: a, 0.01; b, 0.022; c, <0.001; d, 0.0019). H, Newtons;

FIG. 7 shows that AR-42 protects against cancer-induced muscle loss in a mouse model of cachexia LLC. Shown are the approximate effects of AR-42 compared to the carrier in relation to the muscle mass of the hind limbs, including the gastrocnemius, anterior tibial and quadriceps, in mice without tumors and with tumors, compared with mice with tumors receiving the carrier. Mice were treated as described in FIG. 1A, except that mice were killed on day 20 after injection of the tumor cells. Data are presented as mean ± S.D. (n = 8);

FIG. 8 shows exemplary primer sequences used for real-time RT-PCR;

FIG. 9 shows an example path analysis (Ingenuity Pathway Analysis) (IPA) (QIAGEN) of differentially expressed genes (> 4-fold) for muscular diseases or functions between mice with C-26 tumors treated with AR-42 and vehicle (n = 6) ;

FIG. 10 shows an exemplary analysis of cytokine profiles from mice without tumors and mice with C-26 tumors receiving vehicle or AR-42 (mean ± S.D .; n = 3 for each group); and

FIG. Figure 11 shows an exemplary analysis of RNA-seq differentially expressed genes (> 4-times) in muscles from C-26 mice treated with AR-42 or carrier (n = 3).

DETAILED DESCRIPTION OF THE INVENTION

Prior to describing several exemplary aspects presented in the present description, it is necessary to understand that the invention is not limited to the details of the design or process steps indicated in the following description. The aspects described in the present description may be carried out or carried out in various ways.

The aspects presented in this description describe the effects of oral AR-42 in alleviating cachexia-induced weight loss and skeletal muscle atrophy and lengthening survival in a mouse colon cancer cachexia CD2Fi model 26 (C-26). The mouse model of cancer cachexia CD2Fi is described, for example, in BMC Cancer. 2010 Jul 8; 10: 363, incorporated by reference in its entirety. The observed effect of anti-cachexia was associated with the ability of AR-42 to reprogram cell metabolism and suppress IL-6 levels in affected muscle tissue with suppression of muscle loss and other effects of cachexia, regardless of the effects of AR-42 on reducing tumor burden.

Aspects presented in this description describe the effects of oral AR-42 in relation to the induction of cachexia-induced weight loss, skeletal muscle atrophy and lengthening survival in a cancer cachexia model for Lewis lung carcinoma (LLC). See, for example, Expert Opin Drug Discov. Nov 1, 2009; 4 (11): 1145-1155.

The HDAC inhibitors described in US patent number 8318808, can be used in various methods described in the present description. Such HDAC inhibitors are based, for example, on fatty acids bound to Zn ²⁺ chelating moieties via aromatic Ω-amino acid linkers. In various aspects, HDAC inhibitors may have the formula:

where X is selected from H and CH3; Y is (CH2) n, where n is 0-2; Z is selected from (CH2) m, where m is 0-3 and (CH) 2; A is a hydrocarbyl group; B represents an o-aminophenyl or hydroxyl group; and Q is halogen, hydrogen or methyl.

In another aspect, the methods described herein use AR-42, also known as (S) -N-hydroxy-4- (3-methyl-2-phenylbutanamido) benzamide, which has the following chemical structure:

In another aspect, AR-42 includes salts, solvates, hydrates, anhydrous, co-crystalline, and other crystalline forms and combinations. AR-42 can be formulated in a variety of dosage forms that have increased stability, increased bioavailability, continuous release, and other properties.

In one aspect, HDAC inhibitors are classified as zinc-dependent or nicotinamide adenine dinucleotide (NAD) -dependent (Discov Med 10 (54): 462-470, November 2010) and are divided into four classes with eight subtypes of families based on their HDAC substrate: class I (HDAC 1, 2, 3, and 8); class II (HDAC 4, 5, 6, 7, 9, and 10; class III (sirtuins 1-7 (SIRT)); and class IV (HDAC 11). Ibid. In another aspect, HDAC inhibitors include, without limitation, vorinostat (SAHA) (class I and II inhibitor), depsipeptide class I inhibitor, and AR-42 (class I and IIb inhibitor). See, for example, Strahl, BD and Allis, CD. (2000) Nature 403: 41- 45. Other HDAC inhibitors (for example, trichostatin A or TSA) inhibit HDAC class 1 and class 2. Substrates for HDAC inhibitors vary in classes and subtypes.

In another aspect, the HDAC inhibitor inhibits class 1 and class 2b HDAC. In another aspect, the HDAC inhibitor is AR-42.

In a model of cancer cachexia in a colon (26 C) tumor of a colon, a fragment of a C26 tumor was transplanted into BALB / c isogenic mice, and undifferentiated carcinoma developed in mice. Atrophy of skeletal muscles (measured by muscle strength and fatigue resistance) correlated with the observed biochemical changes and the model was described as "a well-standardized experimental model for the study of cancer cachexia." BMC Cancer. 2010 Jul 8; 10: 363.

In one aspect, methods of suppressing cachexia in a mammal with cancer are provided by administering a mammalian HDAC class 1 and 2b inhibitor in an amount effective to substantially maintain the mass of the mammal compared to a mammal that has not received a class 1 and 2b HDAC inhibitor. In another aspect, the HDAC inhibitor is AR-42.

In yet another aspect, the weight of the mammal does not decrease by more than about 6% after about the first 15 days after treatment with AR-42.

In another aspect, the cancer is selected from the group consisting of pancreatic, colon, head, neck, stomach and esophagus cancers. In another aspect, the mammal is a human.

AR-42 can be administered in an amount of from about 1 mg / kg to about 100 mg / kg of a mammal and administered at least once a day. In another aspect, AR-42 is administered twice a day in an amount of about 50 mg / kg of the mammal.

In another aspect, IL-6 levels were reduced by about 56% compared with a mammal that did not receive AR-42. In another aspect, leukemia inhibitory factor (LIF) levels are reduced by about 88% compared with a mammal that did not receive AR-42. In another aspect, the expression of Atrogin-1 mRNA was restored to the basal level compared with a mammal that did not receive AR-42.

In one aspect, the expression of MuRFl mRNA is restored to basal levels compared to a mammal that did not receive AR-42. In another aspect, the increase in cachexia-induced mRNA IL-6RCC is reduced by about 85% compared with a mammal that did not receive AR-42.

In another aspect, the loss of adipose tissue induced by cachexia is substantially recovered compared to a mammal that did not receive AR-42. In another aspect, cachexia-induced reduction in fiber size of skeletal muscles is restored by AR-42 compared to a mammal that did not receive AR-42.

Also provide methods for maintaining skeletal muscle mass in a mammal with cancer, including administering an HDAC class 1 and 2b inhibitor to the specified mammal in an amount effective to maintain at least about 90% of the skeletal muscle mass of the specified mammal for at least , fifteen days compared with a mammal that did not receive a class 1 and 2b HDAC inhibitor.

In another aspect, methods are provided to prolong the survival of a mammal having cancer, including administering a class 1 and 2b inhibitor to a mammal in an amount effective to substantially prolong the survival of the mammal compared to a mammal that did not receive a class 1 and 2b HDAC inhibitor. In yet another aspect, the mammal survives for at least about 21 days after administration of AR-42 to the mammal.

In another aspect, the administration of AR-42 facilitated cachexia-induced weight loss and skeletal muscle atrophy and prolonged mammalian survival. Without being bound to theory, it is believed that the anti-cachectic effect is associated with the ability of AR-42 to reprogram cell metabolism and suppress IL-6 levels in affected muscle tissue with suppression of muscle loss and other effects associated with cachexia, regardless of the effects of AR-42 on tumor reduction. load.

In one aspect, the anti-cachectic effects of AR-42 were evaluated by a variety of techniques, including a CAT-PCR analysis of the expression of established mediators of muscle atrophy in cancer (for example, Cancer Cell. 2008 Nov 4; 14 (5): 369-81), measuring the level of anti-inflammatory serum cytokine and muscle of the gastrocnemius muscles (Am J Pathol. 2011 Mar; 178 (3): 1059-68), analysis of metabolic profiling (J Cachexia Sarcopenia Muscle. 2013 Jun; 4 (2): 145-55), level measurement free amino acids in muscle tissue (J Cachexia Sarcopenia Muscle. 2013 Jun; 4 (2): 145-55; Am J Physiol Endocrinol Metab. 2007 Feb; 292 (2): E501-12), measuring glycolytic signature Ktichesky muscles by measuring the level of biochemical substances associated with glycolysis (Cachexia Sarcopenia Muscle. 2013 Jun; 4 (2): 145-55), measuring the level of glycogen stores in the tissue of the cachectic muscles (Cell Death Differ. 2012 Oct; 19 (10) : 1698-708), analysis of the metabolism of branched-chain amino acids in cachectic muscles (Int J Biochem Cell Biol. 2013 Oct; 45 (10): 2163-72), and measurement of the level of 2-hydroxybutyrate and ophthalmate in cachectic muscles (PLoS One. 2010 May 28; 5 (5): el0883; Int J Cancer. 2010 Febl; 126 (3 ): 756-63).

HDAC inhibitors, as described in the present description, can be administered to a patient in need of treatment (for example, a patient with cancer and exhibiting symptoms of cachexia). In one aspect, certain cancers are especially associated with cachexia, including, without limitation, pancreatic, stomach, head, neck, and esophagus cancers ("cancers associated with cachexia"). In another aspect, an HDAC class 1, 2b inhibitor (for example, AR-42) is administered to a patient in need of treatment.

As used herein, the term “substantially” refers to “the majority of,” “most,” or at least 50%, 60%, 70%, 80%, and 90% by weight or amount, for example, a mammal that does not have cancer.

The terms “treat,” “reduce,” “suppress,” “inhibit,” “prevent,” or similar terms, as used in the present description, do not necessarily mean 100% or complete treatment or prevention. Moreover, such terms refer to different degrees of treatment or prevention of certain diseases (for example, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or 1%), which is understood in the technical field as useful.

The terms "treatment" or "prevention" also refer to delaying the development of a disease for a period of time or delaying development indefinitely. The term "treatment" or "therapy" refers to administering a drug or treating a patient, or prescribing a drug to a patient (for example, a doctor, nurse, or other medical professional), where the patient or a third party (for example, a caregiver, family member or health care professional) introduces a drug or treatment. The term “effective amount” refers to the amount of a drug or therapy (for example, a class I and IIb inhibitor of HDAC) that heals, reduces, inhibits, inhibits, prevents disease (s) or condition (s) (for example, cachexia) or prolongs survival. mammal with a disease or condition.

The term "prolong" or "prolongation", as used in the present description, refers to the increase in the survival time of a mammal receiving treatment, compared with a mammal that does not receive treatment. In this aspect, "increased survival rate" may refer to an increase in the life span of a mammal by, for example, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90 % of the life span of a mammal that does not have cancer.

Any of the HDAC inhibitors described in the present description can be administered orally, parenterally (w / w, w / m, depot-w / m, sc, and depot-sc.), Sublingual, intranasal (inhalation), intrathecal , topically or rectally. Dosage forms known to those skilled in the art are suitable for the delivery of the HDAC inhibitors described herein.

In one aspect, exemplary HDAC inhibitors are administered in an oral dosage form (eg, pill, capsule, caplet or tablet, etc.) to a patient diagnosed with cancer associated with cachexia (eg, pancreatic, bladder, head and neck cancer).

HDAC inhibitors can be formulated into suitable pharmaceutical preparations, such as tablets, capsules or elixirs for oral administration, or into sterile solutions or suspensions for parenteral administration. The HDAC inhibitors described herein can be formulated into pharmaceutical compositions using techniques and procedures well known in the art.

In one aspect, from about 0.1 to 1000 mg, from about 5 to about 100 mg, or from about 10 to about 50 mg of an HDAC inhibitor (eg, AR-42), or its physiologically acceptable salt or ester, can be mixed with a physiologically acceptable base, carrier, excipient, binder, preservative, stabilizer, flavoring, etc. in a standard dosage form, as is customary in pharmaceutical practice. The amount of active substance in compositions or preparations comprising HDAC inhibitors is such that a suitable dosage in the specified range is obtained.

In another aspect, the compositions can be formulated in a standard dosage form, each dosage form contains from about 1 mg to about 500 mg, or from about 10 mg to about 100 mg of the active ingredient. The term “unit dosage form” refers to physically separate units suitable as standard dosages for patients of humans and other mammals, each unit containing a predetermined amount of active substance calculated to obtain the desired therapeutic effect in association with a suitable pharmaceutical excipient.

In one aspect, one or more HDAC inhibitors are mixed with a suitable pharmaceutically acceptable carrier for the preparation of compositions. When mixing or adding the compound (s), the resulting mixture can be a solution, suspension, emulsion or the like. Liposomal suspensions or any other nanoparticle delivery systems can also be used as pharmaceutically acceptable carriers. They can be obtained in accordance with methods known to those skilled in the art. The form of the resulting mixture depends on a number of factors, including the intended route of administration and the solubility of the compound in the chosen carrier or base. In one aspect, the effective concentration is sufficient to reduce or alleviate at least one symptom of the disease, disorder or condition that is being treated, and can be determined empirically.

In another aspect, AR-42 suppresses muscle loss during cachexia, as shown in models of cachexia in C-26 and LLC tumors. Proinflammatory cytokines, IL-6 and TNF, are the main propechectic factors in two models (31, 32). Analysis of the cytokine profile shows that, while AR-42 has no effect on serum TNF levels in mice with C-26 tumors, it decreases serum IL-6 levels and the intramuscular expression of IL-6RCC mRNA. However, mice with C-26 tumors treated with AR-42 still showed elevated serum IL-6 levels and IL-6RCC mRNA compared to mice without tumors, suggesting that reducing the signal for IL-6 is not only responsible for AR -42-mediated suppression of muscle loss.

Mechanistically, the anti-bacterial effect of AR-42 is unique because HDAC inhibitors valproic acid and trichostatin-A cannot reverse muscle loss in mice with C-26 tumors despite their ability to modulate the myostatin / follistatin axis (33). Similarly, the findings showed that, unlike AR-42, vorinostat and romidepsin were ineffective in alleviating cachexia-induced weight loss in the C-26 model. This discrepancy may be due to the greater ability of AR-42 to inhibit the expression of E3 mRNA ligases atrogin-1 and MuRFl in muscle mice with tumors, which may reflect differences in their respective abilities to modulate global gene expression in skeletal muscle. Recent evidence suggests a mechanistic link between pathological acetylation / expression of transcription factors and muscle loss in the affected muscles, leading to the dysregulation of gene expression associated with cachexia [review: (34)]. It is reported that histone-acetyltransferase p300 / CBP activity differentially regulates the transcriptional activity and nuclear localization of transcription factors of the Foxo family in skeletal muscle (35), and that of class 1 HDAC, especially HDAC1, which plays a critical role in mediating muscle atrophy due to poor nutrition or lack of muscle utilization as a result of regulation of expression of Foxo and its targets, atrogin-1 and MuRFl (22).

RNA-seq analysis revealed the ability of AR-42 to reverse a tumor-induced gene expression shift. A total of 677 genes were identified that were differentially expressed 4-fold or more when comparing mice with tumors treated with AR-42 and vehicle. It is possible that such a large number of differentially expressed genes may arise as a result of the action of AR-42 on transcriptional activity and / or expression of a multitude of transcription factors / regulators. In addition to Foxol, AR-42 also modulates the expression of many other transcription factors / regulators, including C / EBPδ, Fos, Jun-b, DAXX, ERN1, HIF3a, MAFF, MAFK, and Mef2c (Fig. 11). Among these transcription factors, the importance of Mef2c in the development of skeletal, cardiac, and smooth muscles is well documented (36), and the AP-1 signaling cascade was involved in cancer-related muscle loss (37).

It was suggested that the cachectic muscles in mice with C-26 tumors demonstrated the physiology of the Warburg tumor, characterized by a high glycolysis rate (38). Metabolic data revealed a pronounced reprogramming of skeletal muscle metabolism in mice with C-26 tumors, which was fully addressed by AR-42. Moreover, the suppressive effect of AR-42 on the production of 2-hydroxybutyrate and ophthalmate, biomarkers of insulin resistance (17) and oxidative stress (18), is remarkable, as substantial evidence associated with insulin resistance (39, 40) and oxidative stress (41) with cachexia.

Mechanistically, the ability of AR-42 to maintain the integrity of skeletal muscles in mice with tumors results from its separate, cumulative effects on changes induced by the tumor in a variety of transcriptional programs and the metabolic phenotype. It is therapeutically important that oral administration of AR-42 at a late stage of tumor growth was still effective in slowing the progression of muscle loss in mice with C-26 tumors. Together, the results show that HDAC inhibitors (for example, AR-42) can be used as part of an effective therapeutic strategy for cancer cachexia, as described in the present description.

Pharmaceutical carriers or bases suitable for administration of HDAC inhibitors described herein include any such carriers suitable for a particular route of administration. In addition, the active substances can also be mixed with other active substances that do not violate the desired action, or substances that complement the desired action or have a different effect. The compounds may be formulated as the sole pharmaceutically active ingredient in the composition or may be mixed with other active ingredients.

In another aspect, if the HDAC inhibitors exhibit insufficient solubility, methods for increasing the solubility may be used. Such methods are known and include, without limitation, the use of co-solvents such as dimethyl sulfoxide (DMSO), the use of surfactants such as TWEEN, and dissolution in aqueous sodium bicarbonate. Derivatives of compounds, such as salts or prodrugs, may also be used in the formulation of effective pharmaceutical compositions.

The concentration of the compound is effective to deliver the amount when administered, which reduces or alleviates at least one symptom of the disease for which the compound is administered. Typically, compositions are formulated for a single dose.

In another aspect, the HDAC inhibitors described in the present description, can be obtained with carriers that protect them from rapid removal from the body, such as compositions of slow release or shell. Such carriers include controlled release formulations such as, without limitation, microencapsulated delivery systems. The active compound may be included in a pharmaceutically acceptable carrier in an amount sufficient to obtain a therapeutically applicable effect in the absence of undesirable side effects in a patient receiving treatment. A therapeutically effective concentration can be determined empirically by examining compounds in the known in vitro and in vivo model systems for diseases that are treated.

In another aspect, HDAC inhibitors and compositions described herein may be included in multi-dose or single dose containers. The included compounds and compositions can be provided in kits, for example, including constituents that can be combined for use. For example, AR-42 in lyophilized form and a suitable diluent can be provided as separate components for mixing before use. The kit may include AR-42 and a second therapeutic agent for co-administration. AR-42 and the second therapeutic agent can be provided as separate constituent parts. A kit may include a plurality of containers, each container containing one or more standard doses of the compounds described herein. In one aspect, the containers can be adapted for the desired route of administration, including, without limitation, tablets, gel capsules, sustained release capsules, and the like for oral administration; depot products, pre-filled syringes, ampoules, vials and the like for parenteral administration; and patches, pads, creams and the like for topical administration.

The concentration of an exemplary HDAC inhibitor in a pharmaceutical composition will depend on the absorption, inactivation, and excretion rate of the active compound, the dosage regimen and amount administered, as well as other factors known to those skilled in the art.

In another aspect, the active ingredient can be administered once, or it can be divided into a series of smaller doses for administration at time intervals. Understand that the exact dosage and duration of treatment are a function of the disease that is being treated, and can be determined empirically using well-known research protocols or by extrapolation from in vivo or in vitro research data. It should be noted that the concentrations and dosage values may also vary depending on the severity of the condition, which is facilitated. It is also understood that for any particular subject, specific administration patterns need to be adjusted over time in accordance with the individual needs and professional opinion of the person who introduces or observes the administration of the compositions, and the concentration ranges indicated in the present description are only exemplary and are not intended to limitations of the scope or implementation of the claimed compositions.

If oral administration is desired, the compound can be provided in a composition that protects it from the acidic environment in the stomach. For example, the composition may be formulated in an enteric coating which retains its integrity in the stomach and releases the active compound in the intestine. The composition can also be formulated in combination with antacid or another such ingredient.

Oral compositions typically include an inert diluent or edible carrier and can be compressed into tablets or enclosed in gelatin capsules. For the purpose of oral therapeutic administration, the active compound or compounds may be included with adjuvants and used in the form of tablets, capsules or lozenges. Pharmaceutically compatible binders and additive materials may be included as part of the composition.

Tablets, pills, capsules, lozenges and the like can contain any of the following ingredients or compounds of a similar nature: a binder such as, without limitation, tragacanth gum, gum arabic, corn starch or gelatin; an excipient such as microcrystalline cellulose, starch or lactose; a disintegrating agent such as, without limitation, alginic acid and corn starch; a lubricant such as, without limitation, magnesium stearate; glidant, such as, without limitation, colloidal silicon dioxide; a sweetener such as sucrose or saccharin; and a flavoring agent such as peppermint, methyl salicylate, or fruit flavors.

When the unit dosage form is a capsule, it may contain, in addition to a material of the above type, a liquid carrier such as fatty oil. In addition, standard dosage forms may contain various other materials that modify the physical form of the dosage form, for example, sugar coatings and other enteric agents. The compounds can also be administered as a component of an elixir, syrup, wafer, chewing gum and the like. A syrup may contain, in addition to the active compounds, sucrose as a sweetener and certain colorants, pigments and dyes, and flavors.

Active materials can also be mixed with other active materials that do not violate the desired action, or with materials that complement the desired action. HDAC inhibitors can be used, for example, in combination with an antitumor substance, hormone, steroid, or retinoid. The antitumor agent may be one of a variety of chemotherapeutic agents, such as an alkylating agent, an antimetabolite, a hormonal agent, an antibiotic, colchicine, Vinca alkaloid, L-asparaginase, procarbazine, hydroxyurea, mitotane, nitrosourea or imidazole carboxamide. Suitable agents include those that provide tubulin depolarization. Examples include colchicine and vinca alkaloids, including vinblastine and vincristine.

In another aspect, the HDAC inhibitors described herein can be co-administered or administered before or after immunizing a patient with a vaccine to enhance the immune response to the vaccine. In one aspect, the vaccine is a DNA vaccine, for example, an HPV vaccine.

In one aspect, solutions or suspensions used for parenteral, intradermal, subcutaneous or topical administration may include any of the following components: a sterile diluent, such as water for injection, saline, non-volatile oil, natural vegetable oils, such as sesame oil, coconut an oil, peanut oil, cottonseed oil and the like, or a synthetic fatty carrier such as ethyl oleate and the like, polyethylene glycol, glycerin, propylene glycol or other synthetic solvents; antimicrobial agents such as benzyl alcohol and methyl parabens; antioxidants such as ascorbic acid and sodium bisulfite; complexing agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates and phosphates; and agents for regulating tone, such as sodium chloride and dextrose. Parenteral preparations may be enclosed in ampoules, disposable syringes, or multi-dose vials made of glass, plastic, or other suitable material. Buffers, preservatives and the like can be included on request.

When administered intravenously, suitable carriers include, without limitation, physiological saline, phosphate buffered saline (PBS), and solutions containing thickeners and solvent agents such as glucose, polyethylene glycol, polypropylene glycol, and mixtures thereof. Liposomal suspensions or any other nanoparticle delivery system, including tissue-specific liposomes, may also be suitable as pharmaceutically acceptable carriers. They can be obtained in accordance with methods known in the art.

In another aspect, HDAC inhibitors can be obtained with carriers that protect the compound from rapid elimination from the body, such as delayed release formulations or coatings. Such carriers include controlled release compositions, such as, without limitation, implants and microencapsulated delivery systems, and biodegradable, biocompatible polymers, such as collagen, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid, and the like. Methods for making such compositions are known to those skilled in the art.

In another aspect, the compounds used in the methods according to the description, can be entered enterally or parenterally. When administered orally, the compounds used in the methods of the present disclosure may be administered in conventional dosage forms for oral administration, as is well known to those skilled in the art. Such dosage forms include the usual solid dosage forms of tablets and capsules, as well as liquid dosage forms such as solutions, suspensions, and elixirs. When solid dosage forms are used, they may be of a sustained release type, so that the compounds used in the methods described in the present description need to be administered only once or twice a day.

Oral dosage forms can be administered to a patient 1, 2, 3, or 4 times per day. The HDAC inhibitors described in the present description, you can enter or three or less times, or even once or twice a day. Therefore, the compounds of HDAC inhibitors used in the methods of the present description, is administered in oral dosage form. Whatever oral dosage form is used, they can be designed to protect the compounds used in the methods described in the present description from the acidic contents of the stomach. Tablets in the enteric coating are well known to those skilled in the art. In addition, capsules filled with small spheres, each coated to protect them from the acidic stomach, are also well known to those skilled in the art.

The terms "therapeutically effective amount" and "therapeutically effective time period" are used to mean treatment at dosages and for periods of time effective to reduce the growth of neoplastic cells. As noted above, such administration may be parenteral, oral, sublingual, transdermal, topical, intranasal, or intrarectal. In one aspect, upon systemic administration, the therapeutic composition can be administered in sufficient dosage to achieve a blood level of compounds of from about 0.1 μM to about 100 mM. For localized administration, substantially lower concentrations than such may be effective, and substantially larger concentrations may be tolerable. A person skilled in the art understands that such a therapeutic effect, resulting in a lower effective concentration of an inhibitor of HDAC or AR-42, can vary significantly depending on the tissue, organ and a particular animal or patient being treated. It is also understood that while a patient may begin with a single dose, such a dose may vary over time if the patient’s condition changes.

It will be apparent to those skilled in the art that the exact dosage and frequency of administration will depend on the particular compounds used in the methods of the description used, the particular condition being treated, the severity of the condition being treated, age, weight and other drugs that the patient can take as well known to the prescribing physician, who is a specialist in the field of technology.

EXAMPLES

The following non-limiting examples illustrate aspects described in the present description.

Example 1

Cancer cachexia models

Model C-26

In one aspect, tumors are obtained by subcutaneous injection of C-26 cells (0.5 × 10 6 cells in 0.1 ml) into the right flank of male CD2F1 mice (approximately 6 weeks old; Harlan Laboratories, Indianapolis, IN) (11). Mice with tumors, as well as mice without tumors serving as controls without cachexia, were randomized to groups that were treated with or AR-42 (50 mg / kg, b.p. through a probe, every other day; Arno Therapeutics, Inc., Flemington , NJ) or carrier (0.5% methylcellulose (w / v) and 0.1% Tween-80 (v / v) in sterile water), starting 6 days after injection of the cells. To evaluate the effect of delayed treatment, treatment with the drug and / or carrier was started 6, 10 and 12 days after injection of the cancer cells.

In another aspect, the effects of AR-42 were compared with other HDAC inhibitors. In this aspect, additional groups of mice with C-26 tumors were treated with vorinostat (50 mg / kg, bp, once a day) and romidepsin (0.6 mg / kg; i.p., twice a week) ( ChemieTek (Indianapolis, IN)).

Model LLC

In another aspect, subcutaneous tumors were obtained from male C57BL / 6 mice (approximately 6 weeks old; Harlan) by injection of 0.5 × 106 LLC cells in the right flank. Treatment with AR-42 and vehicle was performed as for model C-26, starting from day 6 after cell injection. In both models, body weight and food consumption were monitored daily and the size of the tumors was measured at least every two days. Mice were fasted for 2 hours until killing, when the muscles of the forepaws, heart, spleen, epididymal fat and blood were taken and the mass of solid tissues was measured. Muscle samples were frozen in 2-methylbutane, cooled with liquid nitrogen, and then stored at -80 ° C until analysis.

Example 2

Compression force measurement

The force of compression of the forepaw was measured in mice using a digital compression force meter (Columbus Instruments, Columbus, OH). Five measurements were obtained from each mouse, the average of which was designated as the compression force of the mouse.

Morphometric analysis of the size of muscle fibers

Ten-μm sections were cut from frozen skeletal muscle samples using a cryostat (Leica) and then stained with H & E. Images were obtained using an Olympus BX51 microscope (Olympus America, Inc.) and muscle fiber cross-sectional areas were determined using Olympus CellSens software 1.11. Measurements were obtained from five different muscle sections from each of five mice from each group.

RNA isolation, CAT-PCR, and RNA-seq analysis

Total RNA was isolated from homogenized gastrocnemius muscles (n = 3 / group) with the TRIzol reagent (Life Technologies, Carlsbad, CA) and then purified using the RNAeasy Mini Kit (Qiagen, Valencia, CA). kOT-PCR was performed as previously described (42) using a Bio-Rad CFX96 real-time PCR detection system with iQ SYBR Green Supermix (Bio-Rad, Hercules, CA). The primer sequences are listed in FIG. 8. The creation of the RNA-seq library and data analysis was carried out at The Ohio State University Comprehensive Cancer Center (OSUCCC) Nucleic Acid Shared Resource.

Metabolomic and cytokine profile

The calf muscles and serum were collected on day 17 after cell injection for each treatment group (n = 8 / group). Muscles were subjected to Metabolon, Inc. (Durham, NC) for metabolic analysis of 270 metabolic intermediates through its own developed mass spectrometry platform. Serum was subjected to Eve Technologies (Alberta, Canada) to analyze 32 cytokines using a mouse cytokine kit (32-plex panel).

Statistical analysis

Data analysis was performed using SAS 9.3 software (SAS, Inc.; Cary, NC). For experiments with repeated measurements, the data were analyzed by a model of mixed effects, including observational dependencies for each subject. For other experiments involving independent groups, the data were analyzed by ANOVA. For time-to-event type experiments (Fig. 2B), differences in survival functions were compared using a log-rank test. The sets were balanced by the Holmes method for regulating the overall group error probability at 0.05. RNA-seq data was analyzed using the Ingenuity Pathway Analysis (IPA) software (Ingenuity Systems, Redwood City, CA). Only genes with> 4-fold change and P <0.05 were chosen for path analysis.

Example 3

AR-42 suppresses cancer cachexia in the C-26 colon adenocarcinoma model

In one aspect, mice were orally treated with an AR-42 probe (50 mg / kg) or vehicle every other day, starting 6 days after the injection of C-26 cells, when palpable tumors were formed. While the carrier group showed a large decrease in body weight, starting at 12 days, mice treated with AR-42 maintained their weight at a level comparable to that of the control without tumors (Fig. 1A, left). To the end point of the study (day 15), the amount of mass loss, after subtracting the mass of tumors (1 cm ³ volume = 1 gram of mass), reached> 20% for the group receiving the vehicle, and 6% for mice receiving AR-42 (center ). This effect could not be due to a decrease in tumor load, since AR-42 did not alter tumor growth relative to the carrier (right), or increased food intake, since the average daily food intake was comparable in the groups receiving AR-42 and the carrier, and less than that of mice without tumors (Fig. 1C). The mice that received AR-42, despite the large tumor burden, were alive, responsive, active, did not slouch, and did not have uneven wool cover observed in the host-treated relatives at the end point of the present study (Fig. 1B).

Example 4

AR-42 protects muscles from cachexia-induced atrophy

In accordance with the preservation of body weight, skeletal muscle mass was maintained in mice with tumors treated with AR-42. It is indicative of cachexia, the mass of the gastrocnemius, anterior tibial and quadriceps muscles from mice with tumors receiving a carrier (with tumors / carrier) of mice decreased by 20.6%, 10.5%, and 18.1%, respectively, relative to the corresponding muscles from control mice without tumors, whereas those in mice with tumors treated with AR-42 (with tumors / AR-42) mice decreased by 9.6%, 0.8%, and 5.8%, respectively (Fig. 1D ).

Mice with tumors / carrier showed all signs of cachexia, including a significant loss of cardiac and, especially, adipose tissue mass (29.3 ± 6.0% control without tumors), which was facilitated by AR-42 treatment (Fig. 1E, above). Interestingly, AR-42 reliably reduced the mass of adipose tissue by approximately 50% in mice without tumors, while also restoring the loss of adipose tissue mass in mice with tumors to a level compatible with that of mice without tumors / AR-42, the dichotomous effect suggests its ability maintain lipid homeostasis.

In another aspect, mice with C-26 tumors had significantly enlarged spleens, relative to control mice without tumors (11) that did not improve with AR-42 (Fig. 1E, bottom). Since splenomegaly in mice with C-26 tumors resulted from the expansion of myeloid suppressor cells and other immune cells in the spleen (12), this finding suggests that AR-42 acted on muscles rather than through an immunological mechanism.

The protective effect of AR-42 on muscle loss was manifested by the elimination of a cachexia-induced decrease in the size of skeletal muscle fibers. Mice with tumors / carrier showed a decrease of 48.2% relative to the control without tumors in the average sectional area of muscle fibers on day 15 (1297.6 ± 638.8 compared to 2503.5 ± 917.5 μm ² ), which was restored by AR -42 (2146.3 ± 923.4 μm ² ) (Fig. 2A, left). A noticeable shift in the size distribution of the fibers to a smaller cross-sectional area in the cachectic muscles of mice with tumors / carrier was addressed by AR-42 (Fig. 2A, right)

AR-42 lengthens the survival of mice with C-26 tumors.

The effects of AR-42 were compared to other HDAC inhibitors (i.e., vorinostat and romidepsin) in mice with C-26 tumors. Starting from the 6th day after the injection, mice were treated with a long-term AR-42 (50 mg / kg every other day, orally), vorinostat [50 mg / kg once a day, orally (13)], romidepsin [0.6 mg / kg twice a week, ip (14)], or carrier, before weight loss, which was determined by daily determination of body weight and subtraction of the tumor mass, reaching 20% of the initial mass. As shown, oral AR-42 was effective in protecting these mice from emaciation associated with the tumor, with 100% cumulative survival on day 21, when the tumor volume reached the threshold for euthanasia (Fig. 2B). On the contrary, vorinostat and romidepsin showed limited or did not show a noticeable effect on body weight. Moreover, mice with tumors / AR-42 were brisk, responsive, active, and appeared healthy 21 days after injection of the tumor cells, in contrast to mice that were injected with vehicle (day 15), romidepsin (day 16), and vorinostat (day 18 ) (Fig. 2C).

Example 3

Various effects on the regulation of skeletal muscle protein metabolism

The mass of skeletal muscles is regulated by a balance between synthesis and protein breakdown. Without being bound by theory, it is believed that the various anti-cachectic effects of AR-42 compared to vorinostat and romidepsin may be due to differences in their ability to regulate the pathways controlling protein metabolism. This is supported by the suppressive effect of AR-42 on the expression of atrogin-1 / MAFbx mRNA and, MuRFl, two E3 ligases involved in the destruction of ubiquitin-mediated skeletal muscle protein (15, 16) (Fig. 2D).

qPCR analysis of the calf muscles revealed a significant increase in atrogin-1 and MuRFl mRNA levels (29.4 ± 3.5-fold and 25.8 ± 3.9-fold, respectively) in the cachectic muscles (with tumors / carrier; n = 8) relative to control without tumors / carrier (n = 6). AR-42 was able to restore the expression of Atrogin-1 mRNA (2.7 ± 0.7 times) and MuRF1 (1.1 ± 0.2 times) to basal levels (n = 8). Vorinostat (n = 8) and romidepsin (n = 5) also significantly reduced mRNA expression of these two E3 ligases in the cachectic muscles, but to a lesser extent than AR-42 (atrogin-l / MuRFl: vorinostat, 9.6 ± 1, 8 / 5.5 ± 1.1 times; romidepsin, 19.6 ± 3, l / 14.6 ± 3.3 times) (Fig. 2D).

To confirm that the anti-cachectic activity of AR-42 was non-specific for the C-26 model, it was also evaluated in the LLC model. Mice with C57BL / 6 tumors bearing subcutaneous LLC tumors were treated with AR-42 (50 mg / kg bp, every other day), starting on day 6 after the injection of tumor cells, and continued until day 20, when the forelimb muscles were obtained at killing. As shown in FIG. 7, AR-42 defended C57B1 / 6 mice with LLC's tumors from loss of muscle mass (gastrocnemius: vehicle, 81.7 ± 3.7% of non-nichectic control; AR-42, 92.2 ± 3.5%; anterior tibial: carrier , 80.3 ± 4.0%; AR-42, 93.4 ± 3.9%; four-headed: carrier, 84.4 ± 4.6%; AR-42, 93.4 ± 4.8%; all P values <0.05, n = 8).

Example 4

AR-42 maintains the metabolic integrity of the muscles in mice with tumors.

In cachexia, the skeletal muscles undergo complex metabolic changes in response to inflammatory and neuroendocrine stress factors of the tumor and the host organism (1). Accordingly, the authors analyzed the metabolic profile to investigate the effect of AR-42 on cachexia-induced changes in the metabolic phenotype of skeletal muscles. Mice without tumors and with C-26 tumors were treated with vehicle or AR-42, as mentioned above, and the gastrocnemius muscles were taken on day 17 for metabolic analysis. A comparison of muscle biochemical profiles among the four groups (n = 8 / group) revealed the ability of AR-42 to restore cachexia-induced metabolic changes in skeletal muscles, which are summarized below.

Glycolysis . Cachectic muscles from mice with tumors / carrier showed significantly lower levels of glucose and key glycolysis intermediates than control without a tumor (Fig. 3A). AR-42 reversed these metabolic changes by restoring the intramuscular level of glucose and intermediates to, in some cases, and above the initial level determined in mice without tumors / carrier. Moreover, the increased glucose went into the biosynthesis of sorbitol-fructose and the pentose phosphate pathway, leading to increased production of sorbitol, fructose and ribose, a metabolite originating from the pentose phosphate pathway.

Glycogen stores . Muscles from tumors / carrier mice showed a significant decrease in short-chain malto-oligosaccharides and glucose-1-phosphate (Fig. 3B), suggesting depletion of glycogen stores in the cachectic muscles. AR-42 treatment reliably replenished these intermediates of glycogen metabolism.

Free amino acids . In accordance with the increased destruction of the protein, which characterizes muscle loss, a large amount of free amino acids was significantly increased in the cachectic muscles from mice with tumors / carrier, relative to those in mice without tumors / carrier (Fig. 4), demonstrating a cachectic phenotype. Similarly, several amino acid derivatives / metabolites that act as neutrotransmitters, including kinurenin, N-acetyl-aspartylglutamate and γ-aminobutyrate, have been enhanced. On the contrary, alanine, which is released from the muscles to maintain liver gluconeogenesis, declined in the cachectic muscles. Such a cachectic phenotype was treated with AR-42, showing its ability to block the degradation of muscle protein.

Organic acids. Amino acid metabolites 2-hydroxybutyrate and ophthalmate are biomarkers of insulin resistance (17) and oxidative stress (18), respectively. An increase in these two organic acids in the muscles of mice with tumors / carrier (Fig. 4) suggests that cachexia triggers insulin resistance and oxidative stress, which, in turn, exacerbate muscle loss. AR-42 dramatically reduced both biomarkers to a level comparable to levels comparable to those measured in mice without tumors.

Example 5

AR-42 suppresses cancer cachexia by targeting a variety of pro-cachexia trigger factors.

To illuminate the mechanism by which AR-42 carries out its anti-cancer effect, serum and calf muscles from mice without tumors and with C-26 tumors receiving vehicle or AR-42 were used to analyze the cytokine profile and complete sequencing of the short transcriptome (RNA-seq ), respectively.

Cytokine profiles. Of the 32 studied cytokines (Fig. 10), IL-6 and the leukemia inhibitory factor (LIF), two well-known trigger factors for cachexia (19) were significantly elevated in the serum of mice with tumors / carrier relative to that of mice without tumors / carrier ( IL-6; 230 ± 105 relative to 2.9 ± 1.3 pg / ml; LIF, 19.7 ± 9.3 relative to 1.7 ± 1.5 pg / ml) (Fig. 5A, above), while no significant differences were noted for other cytokines. AR-42 reduced IL-6 and LIF levels by 56% and 88%, respectively, in mice with tumors (IL-6, 102 ± 38 pg / ml; LIF, 3.8 ± 1.6 pg / ml) compared with other receiving media. In light of the ability of AR-42 to dull the increase in IL-6 associated with cachexia, the authors investigated the effect of AR-42 on the intramuscular level of the IL-6 receptor alpha chain (IL-6Rα) mRNA. IL-6Rα mRNA was significantly increased (13 ± 1.4-fold) in mice with tumors / vehicle (n = 9) compared with mice without tumors (n = 6). AR-42 reduced such a cachexia-induced increase by 85% (2.0 ± 0.2-fold; n = 10) (Fig. 5, bottom). Such findings suggest that AR-42 inhibits muscle loss, in part, by blocking IL-6 signaling.

RNA seq analysis . The analysis of the main components of RNA-seq data revealed a noticeable effect of C-26 tumor on the variant of global gene expression in muscle mice with tumors / carrier relative to relatives without tumors / carrier (Fig. 5B, left). While AR-42 did not have a noticeable effect on the variant expression of genes in non -tactic muscles without tumors, he reversed the tumor-induced shift in gene expression in the cachectic muscles to a state close to that in mice without tumors. In line with this, the authors performed pairwise analysis of differentially expressed genes between mice with tumors / carrier and the other three treatment groups, the results of which are shown in the Venn diagram (Fig. 5B, right). The widely overlapping areas between paired analyzes suggest that AR-42 restores, to a large extent, tumor-induced changes in global gene expression.

A pairwise comparison of gene expression in muscles from mice with tumors receiving vehicle and AR-42, revealed a total of 677 genes with 4-fold or greater differential expression (376 stimulated and 301 suppressed) (Fig. 10). Analysis of such genomic data in relation to their functional and pathological associations using Ingenuity Pathway Analysis (IPA) revealed that 66 of these differentially expressed genes were attributed to the categories of atrophy, contractility, development and muscular morphology and size of skeletal muscle cells, muscle cell death and protein catabolism (Fig. 11).

Of these genes associated with muscle function and pathology, the effects of AR-42 on the next six genes were noticeable in light of their demonstrated association with cancer-induced cachexia. These include Foxo1 (encoding Forkhead 01 transformer protein) (20-23) and its target genes Trim63 (MuRFl) and Fbxo32 (Atrogin-1) (24, 25), PNPLA2 [adipose triglyceride lipase (ATGL)] (26, 27) , UCP3 (non-binding protein 3) (28, 29), and Mef2c [a factor enhancing myogenic transcription of myocyte factor] (30) (Fig. 5C). Confirmation of RNA-seq data for these six genes by kRT-PCR revealed a high correlation between data groups for relative levels of mRNA expression among the four treatment groups (Fig. 5D).

Example 6

Deferred AR-42 treatment remains effective in suppressing muscle loss.

The above findings demonstrate the effectiveness of oral AR-42 in suppressing muscle loss associated with cancer by restoring metabolic and gene expression profiles in skeletal muscle. In the experiments performed, treatment was started early with the progression of cachexia, when clear signs of loss were undetectable. To investigate whether a later start of AR-42 treatment remains protective against cachexia, mice with C-26 tumors were treated with AR-42 (50 mg / kg, b.a., every other day), starting 6, 10 and 12 days after tumor cell injections.

In accordance with earlier data (Fig. 1), mice with tumors / carrier lost 19% of their body weight (with the exception of the tumor) by 17 days. Conversely, AR-42 treatment, starting at day 6 (D6), 10 (D10), or 12 (D12), limited weight loss to 6%, 11%, and 12%, respectively (n = 8) (Fig. 6A, left), with no noticeable effects on tumor growth (right). Moreover, mice treated with AR-42 showed signs of better health than their relatives treated with the vehicle (Fig. 6B). This protective effect of AR-42 was reflected in the preservation of the mass of the gastrocnemius muscle and to a lesser extent, that of the anterior tibial and quadriceps muscles (Fig. 6C). In accordance with the protective effect on muscle mass, the hand-held dynamometer showed that the AR-42 helped maintain the muscle strength of the forelimbs relative to the control that received the vehicle on days 15 and 16 (Fig. 6D).

Example 7

Mice with tumors / carrier showed other signs of cachexia, including significant loss of cardiac and, especially, adipose tissue mass (29.3 ± 6.0% control without tumor), which was facilitated by AR-42 treatment (Fig. 1E, above). Interestingly, AR-42 reliably reduced adipose tissue mass by approximately 50% in mice without tumors, while still restoring the loss of adipose tissue mass in mice with tumors to a level comparable to that of mice without tumors / AR-42, the dual effect suggests its ability to maintain lipid homeostasis.

Mice with C-26 tumors had extremely enlarged spleens relative to control mice without tumors (11), which did not improve with AR-42 (Fig. 1E, bottom). Since splenomegaly in mice with C-26 tumors resulted from the expansion of myeloid suppressor cells and other immune cells in the spleen (12), this finding suggests that AR-42 acts primarily on the muscles rather than through immunological metabolism.

The protective effect of AR-42 against muscle loss was manifested in the elimination of cachexia-induced reduction in the size of skeletal muscle fibers. Mice with tumors / carrier exhibited a 48.2% decrease relative to control without tumors in the average cross-sectional area of muscle fibers on day 15 (1297.6 ± 638.8 relative to 2503.5 ± 917.5 μm ² ), which was reconstructed by AR- 42 (2146.3 ± 923.4 μm ² ) (Fig. 2A, left). A noticeable shift in the size distribution of the fibers to a smaller cross-sectional area in the cachectic muscles of mice with tumors / carrier was reversed by means of AR-42 (Fig. 2A, right).

Example 8

Differential effects on the regulation of skeletal muscle protein metabolism

Since the skeletal muscle mass is regulated by a balance between protein synthesis and destruction, the differential antikachektichesky effect of AR-42 relative to vorinostat and romidepsin may be due to differences in their ability to regulate the pathways that control protein metabolism. This was supported by the suppressive Atrogin-1 / MAFbx and, MuRFl, two E3 ligases, the implicated and ubiquitin-mediated skeletal muscle protein degradation (15, 16) (Fig. 2D). As expected, qPCR analysis of the calf muscles revealed a significant increase in Atrogin-1 and MuRFl levels (with tumors / carrier; n = 8) relative to the control without tumors / carrier (n = 6). AR-42 was able to restore the expression of atrogin-1 (2.7 ± 0.7 times) and MuRFl (1.1 ± 0.2 times) mRNA to the basal level (n = 8). Vorinostat (n = 8) and romidepsin (n = 5) also significantly reduced mRNA expression of these two ligases in the cachectic muscles, but to a lesser extent than AR-42 (atrogin-1 / MuRF1: vorinostat, 9.6 ± 1.8 / 5.5 ± 1.1 times; romidepsin, 19.6 ± 3.1 / 14.6 ± 3.3 times) (Fig. 2D).

Example 9

Cells

Cultured C-26 and LLC cells were incubated in fetal bovine serum, (FBS) supplemented (10%) RPMI 1640 medium and DMEM medium (Invitrogen, Carlsbad, CA), respectively, at 37 ° C in a humidified incubator with 5% CO ₂ . For injection into mice for models of cancer cachexia, cells were collected by trypsinization, precipitated in FBS-supplemented culture medium, and then resuspended in sterile PBS at a concentration of 5x10 ⁶ cells / ml.

Mice

CD2F1 and C57BL / 6 mice were placed in groups under conditions of a constant photoperiod (12-hour light / 12-hour dark), temperature and humidity with access to water and standard nutrition on demand. Mice were briefly anesthetized (isoflurane, 3-4%) during drug administration (AR-42, vorinostat, vehicle) using an oral probe. Food consumption was assessed by weighing food in each cage daily and dividing the daily food reduction by the number of mice in the cage. Tumor volume was calculated from caliper measurements using a standard formula (length x width x π / 6).

Compression force measurement

To measure the force of the compression of the front paw, each mouse was held by the base of the tail and lowered over the instrument until its front paws were pressed on the pedal. Then the mouse was gently lowered horizontally in a straight line from the force gauge before the mouse released the button, and the maximum force applied was recorded. Five measurements were obtained for each mouse, and the average was designated as the mouse compression force.

Creating a library of RNA-seq and data analysis channel

RNA quality was evaluated on an Agilent 2100 bioanalyzer using an RNA template and the incoming total amount of RNA was evaluated using an Agilent Qubit RNA analysis. Libraries of transcriptomes were obtained using an Illumina TruSeq V2 RNA sample kit. The resulting libraries were evaluated for quality and quantity using Agilent Qubit DNA analysis and PerkinElmer Labchip DNA GX analysis, respectively. All libraries were mixed in equal proportions, generating sample pools that produced approximately 40 million missing filter reads when sequenced with the Illumina HiSeq 2500 sequencer. The initial sequencing data from the Illumina HiSeq CASAVA channel was evaluated for quality using FastQC, RNASeQC and RSeQC software. Subsequent analyzes were as follows: read the sequencing of the demultiplex pass filter aligned with GRCm38 / mm10 using the TopHat 2 (v2.0.7) RNAseq setup; CuffLinks 2 (c2.1.1) was used to assemble aligned readings to designate the UCSC gene mm10; CuffCompare and CuffMerge used to compose aligned readings into mm10 genes and sorted the assembled transcripts into the designation of a special gene; CuffDiff was used to compare differential gene expression associated with each treatment group.

Example 10

To confirm that the anti-bacterial activity of AR-42 was non-specific for the C-26 model, it was also evaluated in the LLC model. C57BL / 6 mice bearing subcutaneous 11 LLC tumors were treated with AR-42 (50 mg / kg p.o., every other day) starting at 6 days after the injection of tumor cells, and continuing until the 20th day when the hind paw muscles were received at killing.

As shown in FIG. 7, AR-42 protects against cancer-induced muscle loss in a mouse model of cachexia LLC. The effects of AR-42 relative to the carrier on the muscle mass of the hind limbs, including the gastrocnemius, anterior tibial and quadriceps, in mice without tumors and with tumors were compared to those of mice with tumors receiving the carrier. Mice were treated in the same manner as described in FIG. 1A, except that mice were killed on day 20 after injection of tumor cells. Data are presented as mean ± S.D. (n = 8); (sural: carrier, 81.7 ± 3.7% of non-cachectic control; AR-42, 92.2 ± 3.5%; anterior tibial: carrier, 80.3 ± 4.0%; AR-42, 93.4 ± 3.9%; quadriceps: carrier, 84.4 ± 4.6%; AR-42, 93.4 ± 4.8%; all values are P <0.05, n = 8).

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Although the above description relates to certain aspects, it is necessary to understand that such aspects are merely illustrative. It is obvious to a person skilled in the art that various modifications and variations can be made with the methods described in the present description. Therefore, the present description includes modifications and variations that are within the scope of the appended claims and their equivalents.

Claims (9)

1. A method of reducing cancer-induced cachexia in a mammal, comprising administering an HDAC class 1 and 2b inhibitor to said mammal in an amount effective to preserve the body weight of the mammal, compared with a mammal that does not receive an HDAC inhibitor of class 1 and 2b. 2. A method according to claim 1, wherein the HDAC inhibitor is AR-42. 3. The method according to claim 1, wherein the cancer is selected from the group consisting of pancreatic, colon, head, neck, stomach and esophagus cancers. 4. The method according to claim 1, wherein the mammal is a human. 5. A method according to claim 2, where the AR-42 is administered in an amount of from 1 mg / kg to 100 mg / kg of a mammal. 6. The method according to claim 5, where the AR-42 is administered at least once a day. 7. The method according to claim 6, where the AR-42 is administered twice a day in an amount of 50 mg / kg of a mammal. 8. A method of maintaining skeletal muscle mass in a mammal having cancer, where the skeletal muscle mass is reduced due to cancer, comprising administering a class 1 and 2b HDAC inhibitor to said mammal. 9. The method according to claim 8, wherein the HDAC class 1 and 2b inhibitor is AR-42.

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